Patent Application
20210085736
Mesothelioma is an asbestos-associated malignant form of cancer, which often has a poor prognosis in humans. The current standard of care for this life-threatening malignancy only achieves suboptimal improvements in patient survival. Harnessing the host immune system to eradicate malignant cells has become a clinical strategy in cancer immunotherapy. Although immune checkpoint inhibitors have improved the therapeutic efficacy in certain cancers, their effects are unsatisfactory in patients with mesothelioma. Therefore, novel strategies are needed for treating mesothelioma. Recently, oncolytic virotherapy has emerged as a promising cancer immunotherapy for the treatment of solid tumors including malignant mesothelioma. However, the mechanisms underlying the limited virotherapeutic efficacy remains elusive.
Direct virus-mediated oncolysis of cancer cells is one of the major mechanisms of oncolytic virotherapy. During oncolysis, danger-associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) are released into the tumor microenvironment (TME), which can modulate the immunogenicity of released tumor antigens by creating an immune-activating environment and subsequently eliciting or reinforcing tumor-reactive T cell responses. The crucial role of adaptive T cell immunity in oncolytic virotherapy has been demonstrated in both preclinical and clinical studies. However, TME is often an immunosuppressive environment that inhibits the activation of tumor-reactive T cells by inducing tolerogenic dendritic cells (DCs) and CD25+Foxp3+ regulatory T lymphocytes (Tregs). Bone marrow myeloid-derived suppressor cells (MDSCs) in the TME can dampen the responsiveness of cytotoxic T lymphocytes (CTLs), leading to limited efficacy in patients, especially when the TME is highly immunosuppressive. Because T cell immunity is indispensable for the efficacy of oncolytic virotherapy, the better understanding of restrictive mechanisms in the TME is particularly important for improving the clinical outcomes of oncolytic virotherapies.
MDSCs represent one of the major immunosuppressive populations in the TME and a major obstacle to the effectiveness of cancer immunotherapy. In malignant mesothelioma models, MDSCs expand quickly with the development of tumor lesions and contribute to the inhibition of tumor-reactive CTL responses. Consistently, decreased numbers of MDSCs in the TME are likely associated with the generation of antigen-specific CTL responses and therapeutic efficacy during oncolytic virotherapy in patients. MDSCs can be monocytic (M) or polymophonuclear (PMN). Targeting the COX-2-PGE2 pathway during vaccinia virotherapy is capable of decreasing PMN-MDSC levels while increasing antitumor CTL responses. Moreover, an earlier study using the COX-2 inhibitor celecoxib improved DC-based immunotherapy against mesothelioma by reducing the PMN-MDSC frequency. While these studies indicate the critical role of PMN-MDSCs in cancer immunotherapy, curing established tumors has rarely been observed. To date, the mechanism underlying MDSCs accumulation in the TME, the functional difference between MDSC subsets, and their impact on eliciting antitumor CTLs during oncolytic virotherapy remain incompletely understood.
In certain embodiments, the invention provides methods of treating a cancer in a subject by administering to the subject a combination of an oncolytic virus and a therapy that induces depletion of tumor-induced PMN-MDSCs. In preferred embodiments, the oncolytic virus is a replication incompetent modified vaccinia TianTan (MVTT) virus having a deletion of the viral M1L-K2L genes. In other preferred embodiments, the therapy that induces depletion of tumor-induced PMN-MDSCs comprises administering an antibody against Ly6G, for example 1A8. The cancer therapies of the invention can be administered in combination with one or more additional anti-cancer therapies. Preferred additional anti-cancer therapy is an immunotherapy, such as administering a check-point inhibitor.
Cancer virotherapy using oncolytic viruses is a promising therapeutic strategy with demonstrated clinical benefits. Following the approval of T-vec (also known as Imlygic), a recombinant herpes simplex virus expressing the immune-activating cytokine GM-CSF for treating skin and lymph node melanoma in the USA and Europe, a variety of oncolytic viruses have progressed to clinical development. Among these, the use of ONCOS-102 adenovirus for treating malignant mesothelioma was able to induce tumor-infiltration by CD8+ T cells, systemic antitumor CD8+ T cells and Th1-type polarization in a clinical setting. Although the therapeutic effects of T-vec and ONCOS-102 are promising, only a small fraction of treated patients experienced clinical responses in these studies. Therefore, investigating how to induce potent antitumor immune responses is essential for enhancing the therapeutic efficacy of virotherapy in patients. Most of the viruses that are currently being tested in clinical trials were designed to acquire the capability to trigger immune responses. To this end, understanding the mechanism underlying the blockade and regulation of systemic antitumor immunity is critical for further improvement of oncolytic virotherapy.
Replication of the oncolytic virus in the tumor releases the danger signals CRT, HMGB1 and ATP, as well as tumor antigens for DCs, to trigger antitumor immune responses. Therefore, a combination therapy with an immunotherapy has become a useful strategy to improve the efficacy of oncolytic virotherapy in fighting various types of tumors, including malignant mesothelioma and melanoma. An immunotherapy includes augmenting host antitumor responses through the incorporation of immune activating molecules (e.g., GM-CSF), immune-regulatory drugs (e.g., cyclophosphamide), or immune checkpoint inhibitors.
In addition to the rapidly increased use of immune checkpoint inhibitors, a GM-CSF-incorporated herpes simplex virus (T-vec) has also received regulatory approval for treating patients with late-stage melanoma. Decreasing immune suppression of MDSCs and Tregs by sunitinib has been shown in clinical trials to augment anti-renal cell carcinoma immune responses during oncolytic reovirus treatment. In terms of malignant mesothelioma, the use of first-line chemotherapeutic agents (cisplatin or pemetrexed) during oncolytic adenovirus treatment has been shown to enhance virus-mediated cytotoxicity in mice.
MVTT virotherapy alone is insufficient for efficient tumor clearance. Replication of the oncolytic virus in the tumor releases the danger signals CRT, HMGB1, ATP, and tumor antigens for DCs to trigger antitumor immune responses. However, complete mesothelioma eradication was only achieved by intra-tumoral administration of extremely high doses of MVTT at multiple sites of the solid tumors, yet even in protected mice, antitumor T cell responses were rarely elicited.
The instant invention describes that virotherapy significantly expanded MDSCs in the mesothelioma TME. Expansion of MDSCs is a key immune evasion mechanism in various human cancers, such as renal cell carcinoma, squamous cell carcinoma, breast cancer, and non-small cell lung carcinoma. In mice with mesothelioma, tumors induced a rapid increase of MDSCs as early as 7 days after AB1 cell inoculation and the elimination of MDSCs during immunotherapy was closely related to tumor rejection. Expanded PMN-MDSCs in the mesothelioma TME during MVTT virotherapy were due to the production of C—X—C chemokines associated with the viral infection of tumor cells. C—X—C chemokines then preferentially recruit CXCR2+ PMN-MDSCs from peripheral lymphoid organs to tumor sites by chemotaxis. These results emphasize the role of the C—C and C—X—C axes in the trafficking of M-MDSCs and PMN-MDSCs, respectively.
Viral infection-recruited PMN-MDSCs were found to be responsible for either suppression of NK cells by reactive oxygen species (ROS) production or augmentation of local immune suppression by PD-L1 expression. The instant invention demonstrates that PMN-MDSCs exhibited potent immunosuppressive function against DC activation. Similar immunosuppressive effects on DCs were not found with M-MDSCs, suggesting a functional difference between these two MDSC subsets in the mesothelioma TME.
Depletion of PMN-MDSCs alone is also insufficient for efficient tumor clearance. Targeted depletion of PMN-MDSCs allowed modest CTL responses in pancreatic ductal adenocarcinoma and lung cancer models. AB1 mesothelioma in mice, however, has been recognized as a poorly immunogenic model. AB1 mesothelioma displayed similar growth kinetics in immunodeficient SCID mice compared to immunocompetent BALB/c mice.
Moreover, purified T cells from mesothelioma-bearing mice did not contain antigen-specific T cells with potent cytotoxic activity. To better define the function of PMN-MDSCs and M-MDSCs in modulating antitumor immunity, depletion experiments using anti-Ly6G or H6-pep monotherapy, respectively, were conducted. Depletion of either PMN-MDSCs or M-MDSCs did not induce any inhibitory effects on mesothelioma growth. Additionally, no measurable antitumor CTLs were detected. Therefore, depletion of MDSCs subsets alone did not promote the exposure of mesothelioma antigens to trigger DC activation. Thus, an oncolytic virotherapy is necessary to promote tumor antigen exposure and subsequent induction of systemic antitumor T cell responses.
Thus, the instant invention demonstrates that curing established mesothelioma requires a combination of an oncolytic virotherapy, such as MVTT virotherapy, and PMN-MDSC depletion, which can overcome immunosuppression despite increasing intra-tumoral M-MDSCs and potentiate DCs for the induction of potent antitumor CTLs. PMN-MDSCs play a critical role in modulating antitumor CTL responses. Using the PMN-MDSC-depleting antibody 1A8 and M-MDSC-depleting peptibody H6-pep, PMN-MDSCs but not M-MDSCs are shown to be essential for the TME to restrict the induction of tumor-reactive CTL responses during an oncolytic virotherapy, such as MVTT virotherapy.
Moreover, the combination of an oncolytic virotherapy, such as MVTT virotherapy, and depletion of PMN-MDSCs activated endogenous T cells to elicit antitumor CTLs with broad-reactive spectrum, cytolytic activity, and protective long-term memory responses. During this process, increased intra-tumoral M-MDSCs were unable to block T cell activation and antitumor CTLs.
Mechanistically, intra-tumoral PMN-MDSCs but not M-MDSCs suppressed DC activation by preventing CD80 and CD86 upregulation and IL-6, TNF-α and IL-12p70 secretion. Therefore, in addition to the suppressive effects of MDSCs on T cells, the invention describes the mechanisms by which mesothelioma-derived PMN-MDSCs exhibit immune suppressive activity on DCs. Cross-talk between PMN-MDSCs and DCs demolished antitumor immunity by increasing IL-10 production and decreasing DC activation.
Tumor-derived MDSCs upregulated IL-10 production and neutralization of IL-10 abrogated the suppressive effect of MDSCs in mouse models. Given the plasticity of the immune suppressive myeloid compartment under various tumors and infectious agents, acute phase response protein induced the expansion and polarization of IL-10-secreting tumor associated neutrophils to suppress antigen specific T cell responses in melanoma patients. Thus, IL-10-sereting PMN-MDSCs act as a barricade to protect tumors from immune surveillance. Chemotactically recruited IL-10-sereting PMN-MDSCs are critical DC suppressors to halt T cell activation during the MVTT virotherapy.
Inhibiting cell cycle-related kinase (CCRK) signaling diminished PMN-MDSC mediated immunosuppression and inhibited tumorigenicity of hepatocellular carcinoma. Therefore, an epigenetic modulatory approach targeting CCRK to specifically disrupt PMN-MDSC accumulation would be especially important in the development of combination therapy with MVTT for treating a variety of human cancers, such as mesothelioma.
Thus, the invention describes that intra-tumoral PMN-MDSCs are key suppressors of DC in the mesothelioma TME that restrict the induction of antitumor CTLs, compromising the efficacy of MVTT-based virotherapy.
Accordingly, certain embodiments of the invention provide a method of treating a cancer, such as mesothelioma, by administering a combination of an oncolytic virus and a therapy that induces depletion of tumor-induced PMN-MDSCs.
An oncolytic virus and a therapy that induces depletion of tumor-induced PMN-MDSCs can be administered simultaneously or consecutively. An oncolytic virus can be administered before or after administering a therapy that induces depletion of tumor-induced PMN-MDSCs. Co-administration of an oncolytic virus and a therapy that induces depletion of tumor-induced PMN-MDSCs can be carried out in the same or separate compositions. Separate administrations of these therapies can be performed with one or more additional agents.
When administered separately, an oncolytic virus can be administered within about one day to about seven days, preferably, within about two days to about six days, more preferably within about three to five days, and even more preferably, within about four days of administering a therapy that induces depletion of tumor-induced PMN-MDSCs. In other embodiments, when administered separately, an oncolytic virus can be administered within about 20 to 40 hours, preferably about 25 to 35 hours, even more preferably, about 30 hours, and most preferably, about 24 hours of administering a therapy that induces depletion of tumor-induced PMN-MDSCs.
In preferred embodiments, an oncolytic virus is administered before administering a therapy that induces depletion of tumor-induced PMN-MDSCs.
An oncolytic virus and a therapy that induces depletion of tumor-induced PMN-MDSCs can be administered multiple times over a period of days, for example, over two to fourteen days, more preferably, over four to twelve days, more preferably, over six to ten days, and even more preferably over about seven days.
In some embodiments, the oncolytic virus is an adenovirus, reovirus, herpes virus, picornavirus (including coxsackievirus, poliovirus, and Seneca Valley virus), paramyxovirus (including measles virus and Newcastle disease virus (NDV)), parvovirus, rhabdovirus (e.g., vesicular stomatitis virus (VSV), or vaccinia virus. The oncolytic virus can be replication competent or replication incompetent. Methods of producing replication incompetent viruses are known in the art and are within the purview of the instant invention.
In specific embodiments, the oncolytic virus is a modified vaccinia virus. Preferably, a modified vaccinia virus is a live-attenuated vaccinia virus, such as a vaccinia virus incapable of replication. In some embodiments, modified vaccinia virus is a genetically modified vaccinia virus having a deletion of one or more genes that are necessary for replication. For example, deletion of M1L-K2L genes renders a vaccinia virus incapable of replication.
An example of a modified vaccinia virus, particularly, modified vaccinia TianTan (MVTT) virus, that is suitable for use in the instant invention is described by Zhu et al. (2007), J Virol Methods; 144(1-2):17-26. The Zhu et al. reference is incorporated by reference in its entirety.
In certain embodiments, a modified vaccinia virus is a MVTT generated from vaccinia TianTan (VTT) by deleting the viral M1L-K2L genes. In other embodiments, a modified vaccinia virus is a MVTT generated from VTT by replacing the viral M1L-K2L genes with a heterologous gene, such as a gene encoding a marker fluorescent protein. Compared to the parental VTT, MVTT is 100-fold less virulent. Therefore, MVTT is an attenuated vaccinia Tian Tan vaccine vector with improved safety.
Thus, in specific embodiments, the oncolytic virus is a MVTT.
In further embodiments, the oncolytic virus is a recombinant MVTT (rMVTT). The rMVTT comprises a deletion of the viral M1L-K2L genes from a VTT and further comprises two or more heterologous genes that replace the deleted viral M1L-K2L genes. One of the two or more heterologous genes can be a gene encoding a protein label, such as a fluorescent protein or an enzyme. The fluorescent protein can be a green fluorescent protein or a red fluorescent protein. Red fluorescent protein can be HcRed or green fluorescent protein (GFP). Additional examples of fluorescent proteins are known to a person of ordinary skill in the art and such embodiments are within the purview of the invention. For example, fluorescent protein database (fpbase) is well known in the art and can be found at world-wide-web site: fpbase.org.
In further embodiments, one of the two or more heterologous genes is a gene encoding a capsid protein of a heterologous virus, preferably, p24 protein of human immunodeficiency virus (HIV). The term “heterologous virus” as used herein refers to a virus other than a VTT.
In specific embodiments, one of the two or more heterologous genes is a gene encoding a fluorescent protein and another one of the two or more heterologous genes is a gene encoding a capsid protein of a heterologous virus. Preferably, one of the two or more heterologous genes is a gene encoding HcRed and another one of the two or more heterologous genes is a gene encoding p24 of HIV.
In further embodiments, one of the two or more heterologous genes is under the control of a synapsin promoter (pSYN) and another one of the two or more heterologous genes is under the control of an H5 promoter (pH5). Preferably, one of the two or more heterologous genes is a gene encoding HcRed under the control of pH5 and another one of the two or more heterologous genes is a gene encoding p24 of HIV under the control of pSYN.MVTT readily induces DAMPs including calreticulin (CRT) exposure, HMGB1 and ATP release, as well as oncolysis of AB1 mesothelioma cells. MVTT elicits tumor-reactive CTLs, which are essential for curing malignant mesothelioma. MVTT virotherapy also induces chemotaxis that recruits IL-10-producing PMN-MDSCs into the TME, where they suppress DCs and therefore block the induction of antitumor CTLs. Depletion of PMN-MDSCs but not of M-MDSCs during MVTT virotherapy unleashes tumor-reactive CTLs leading to the therapeutic cure of a cancer, such as mesothelioma. The invention provides that the depletion of MDSCs, particularly PMN-MDSCs, in combination with oncolytic MVTT treatment, can restore potent antitumor T cell immunity, for example, by eliciting cytotoxic CD8+ T cell responses.
Accordingly, specific embodiments of the invention provide a method of treating a cancer, such as malignant mesothelioma or melanoma, by administering a combination of an oncolytic MVTT and a therapy that induces depletion of tumor-induced PMN-MDSCs.
Certain examples of therapies that induce depletion of tumor-induced PMN-MDSCs include gemcitabine, fluorouracil, bindarit, PDE5 inhibitors, tadalafil, nitroaspirin, COX-2 inhibitors, ipilimumab, bevacizumab, celecoxib, sildenafil and tadalafil, N-hydroxy-L-arginine, N-acetyl cysteine (NAC), CpG oligodeoxy-nucleotides (ODN), Bardoxolone methyl (CDDO-Me), withaferin A, Monoclonal anti-Gr1 antibody, IL4Ra aptamer, and peptibodies that target MDSC-membrane proteins (S100 family).
In specific embodiments, therapies that induce depletion of tumor-induced PMN-MDSCs are specific only for inducing depletion of tumor-induced PMN-MDSCs without affecting tumor-induced M-MDSCs.
In preferred embodiments, a therapy that induces depletion of tumor-induced PMN-MDSCs is an antibody against lymphocyte antigen 6 complex locus G6D (Ly6G), for example, antibody 1A8. An antibody against Ly6G, such as 1A8 specifically induces depletion of tumor-induced PMN-MDSCs without affecting tumor-induced M-MDSCs.
In certain embodiments, the methods comprise administering a chemotherapeutic agent before, during, or after administering a combination of an oncolytic virus and a therapy that induces depletion of tumor-induced PMN-MDSCs.
In further embodiments, an irradiation therapy is administered to the subject before or after administering a combination of an oncolytic virus and a therapy that induces depletion of tumor-induced PMN-MDSCs. An irradiation therapy can also be administered between administering an oncolytic virus and administering a therapy that induces depletion of tumor-induced PMN-MDSCs.
In certain embodiments, the methods comprise administering a check-point inhibitor to the subject before, during, or after administering a combination of an oncolytic virus and a therapy that induces depletion of tumor-induced PMN-MDSCs. A check-point inhibitor therapy can be administered between administering an oncolytic virus and administering a therapy that induces depletion of tumor-induced PMN-MDSCs.
Certain checkpoint inhibitors have been used in cancer therapy. Checkpoints refer to inhibitory pathways in the immune system that are responsible for maintaining self-tolerance and modulating the degree of immune system response to minimize peripheral tissue damage. Tumor cells can activate immune system checkpoints to decrease the efficacy of immune response against tumor tissues. Administering checkpoint inhibitors release the inhibition on the immune system and allow immune system activity against the tumor cells. Exemplary checkpoint inhibitors include inhibitors, such as antibodies, against cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152), programmed cell death protein 1 (PD-1, also known as CD279) and programmed cell death 1 ligand 1 (PD-L1, also known as CD274). Exemplary anti-PD-1 antibodies are commercially available and include pembrolizumab, lambrolizumab, nivolumab, AMP-224 (MERCK), and pidilizumab. Exemplary anti-PD-L1 antibodies are also commercially available and include atezolizumab, MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH), BMS-936559 (BRISTOL-MYERS SQUIBB), and AFFYMETRIX EBIOSCIENCE (MIH1). Exemplary anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). Ipilimumab has recently received FDA approval for treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11:89). Additional checkpoint inhibitors are well known to a skilled artisan and such embodiments are within the purview of the invention.
Examples of cancers that can be treated according to the materials and methods disclosed herein include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, cervical cancer, ovarian cancer, peritoneal cancer, liver cancer, e.g., hepatic carcinoma, bladder cancer, colorectal cancer, endometrial carcinoma, kidney cancer, and thyroid cancer. In some embodiments, the cancer is melanoma, MDS, ovarian cancer, breast cancer, or multiple myeloma.
In some embodiments, the cancer is malignant mesothelioma or melanoma.
Other non-limiting examples of cancers are basal cell carcinoma, biliary tract cancer; bone cancer; brain and CNS cancer; choriocarcinoma; connective tissue cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer; lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; sarcoma; skin cancer; stomach cancer; testicular cancer; uterine cancer; cancer of the urinary system, as well as other carcinomas and sarcomas. Examples of cancer types that may be treated with the compositions and methods of the invention are listed in Table 1.
As used herein, the term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. For example, a particular cancer may be characterized by a solid mass tumor or non-solid tumor. The solid tumor mass, if present, may be a primary tumor mass. A primary tumor mass refers to a growth of cancer cells in a tissue resulting from the transformation of a normal cell of that tissue. In most cases, the primary tumor mass is identified by the presence of a cyst, which can be found through visual or palpation methods, or by irregularity in shape, texture or weight of the tissue. However, some primary tumors are not palpable and can be detected only through medical imaging techniques such as X-rays (e.g., mammography) or magnetic resonance imaging (MM), or by needle aspirations. The use of these latter techniques is more common in early detection. Molecular and phenotypic analysis of cancer cells within a tissue can usually be used to confirm if the cancer is endogenous to the tissue or if the lesion is due to metastasis from another site. Some tumors are unresectable (cannot be surgically removed due to, for example the number of metastatic foci or because it is in a surgical danger zone). The treatment and prognostic methods of the invention can be utilized for early, middle, or late stage disease, and acute or chronic disease.
Various methods may be used to deliver to a subject an oncolytic virus and/or a therapy that induces depletion of tumor-induced PMN-MDSCs. The oncolytic virus and the therapy that induces depletion of tumor-induced PMN-MDSCs can both be administered via the same route. Alternatively, the oncolytic virus can be administered via one route and the therapy that induces depletion of tumor-induced PMN-MDSCs can be administered via a different route. In preferred embodiments, the oncolytic virus and the therapy that induces depletion of tumor-induced PMN-MDSCs are both administered i.t.
The oncolytic viruses and the therapy that induces depletion of tumor-induced PMN-MDSC can be administered in one or more pharmaceutical compositions. The pharmaceutical compositions can include various other components. Examples of acceptable components or adjuncts which can be employed used in the pharmaceutical compositions include antioxidants, free radical scavenging agents, peptides, growth factors, antibiotics, bacteriostatic agents, immunosuppressives, anticoagulants, buffering agents, anti-inflammatory agents, anti-angiogenics, anti-pyretics, time-release binders, anesthetics, steroids, and corticosteroids. Such components can provide additional therapeutic benefit, enhance the therapeutic action of the anti-cancer therapy or act towards preventing any potential side effects of the anti-cancer therapy.
Additional agents can be co-administered to subjects or into the cancer cells in a subject in the same or separate formulations. Such additional agents include agents that modify a given biological response, such as immunomodulators. The additional agents may be, for example, small molecules, polypeptides (proteins, peptides, or antibodies or antibody fragments), or nucleic acids (encoding polypeptides or inhibitory nucleic acids such as antisense oligonucleotides or interfering RNA). For example, proteins such as tumor necrosis factor (TNF), interferon (such as alpha-interferon and beta-interferon), nerve growth factor (NGF), platelet derived growth factor (PDGF), and tissue plasminogen activator can be administered. Biological response modifiers, such as lymphokines, interleukins (such as interleukin-1 (IL-1), interleukin-2 (IL-2), and interleukin-6 (IL-6)), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors can be administered. In one embodiment, the methods and compositions of the invention incorporate one or more anti-cancer agents, such as cytotoxic agents, chemotherapeutic agents, anti-signaling agents, and anti-angiogenic agents.
In some embodiments, the compositions of the invention include at least one additional anti-cancer agent (e.g., a chemotherapeutic agent). In some embodiments of the methods of the invention, at least one additional anti-cancer agent is administered with the compositions of the invention. In some embodiments, the anti-cancer agent is selected from among suberoylanilide hydroxamic acid (SAHA) or other histone deacetylase inhibitor, arsenic trioxide, doxorubicin or other anthracycline DNA intercalating agent, and etoposide or other topoisomerase II inhibitor.
In some embodiments, the compositions can include, and the methods can include administering, one or more proteasome inhibitors (e.g., bortezomib), inhibitors of autophagy (e.g., chloroquine), alkylating agents (e.g., melphalan, cyclophosphamide), MEK inhibitors (e.g., PD98509), FAK/PYK2 inhibitors (e.g., PF562271), or EGFR inhibitors (e.g., erlotinib, gefitinib, cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab), or a combination of two or more of the foregoing.
Thus, an oncolytic virus or a therapy that induces depletion of tumor-induced PMN-MDSCs, whether administered separately, or as a pharmaceutical composition, can include various other components as additives. Examples of acceptable components or adjuncts which can be employed in relevant circumstances include antioxidants, free radical scavenging agents, peptides, growth factors, antibiotics, bacteriostatic agents, immunosuppressives, anticoagulants, buffering agents, anti-inflammatory agents, anti-angiogenics, anti-pyretics, time-release binders, anesthetics, steroids, and corticosteroids. Such components can provide additional therapeutic benefit, act to affect the therapeutic action of the compounds of the invention, or act towards preventing any potential side effects which may be posed as a result of administration of the compounds. The immunotherapeutic agent can be conjugated to a therapeutic agent or other agent, as well.
As used herein, the term “immunotherapy” refers to the treatment of disease via the stimulation, induction, subversion, mimicry, enhancement, augmentation or any other modulation of a subject's immune system to elicit or amplify adaptive or innate immunity (actively or passively) against cancerous or otherwise harmful proteins, cells or tissues. Immunotherapies (i.e., immunotherapeutic agents) include cancer vaccines, immunomodulators, monoclonal antibodies (e.g., humanized monoclonal antibodies), immunostimulants, dendritic cells, and viral therapies, whether designed to treat existing cancers or prevent the development of cancers or for use in the adjuvant setting to reduce likelihood of recurrence of cancer. Examples of cancer vaccines include GVAX, Stimuvax, DCVax and other vaccines designed to elicit immune responses to tumor and other antigens including MUC1, NY-ESO-1, MAGE, p53 and others. Examples of immunomodulators include 1MT, Ipilimumab, Tremelimumab and/or any drug designed to de-repress or otherwise modulate cytotoxic or other T cell activity against tumor or other antigens, including, but not restricted to, treatments that modulate T-Reg cell control pathways via CTLA-4, CD80, CD86, MHC, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, CD28, other TCRs, PD-1, PDL-1, CD80, ICOS and their ligands, whether via blockade, agonist or antagonist. Examples of immunostimulants include corticosteroids and any other anti- or pro-inflammatory agent, steroidal or non-steroidal, including, but not restricted to, GM-CSF, interleukins (e.g., IL-2, IL-7, IL-12), cytokines such as the interferons, and others. Examples of dendritic cell (DC) therapies include modified dendritic cells and any other antigen presenting cell, autologous, allogeneic, or xenogeneic, whether modified by multiple antigens, whole cancer cells, single antigens, by mRNA, phage display or any other modification, including but not restricted to ex vivo-generated, antigen-loaded dendritic cells (DCs) to induce antigen-specific T-cell immunity, ex vivo gene-loaded DCs to induce humoral immunity, ex vivo-generated antigen-loaded DCs induce tumor-specific immunity, ex vivo-generated immature DCs to induce tolerance, including but not limited to Provenge and others. Examples of viral therapies include oncolytic viruses or virus-derived genetic or other material designed to elicit anti-tumor immunity and inhibitors of infectious viruses associated with tumor development, such as drugs in the Prophage series. Examples of monoclonal antibodies include Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab ozogamicin, Rituximab, Trastuzumab, Radioimmunotherapy, Ibritumomab tiuxetan, Tositumomab/iodine tositumomab regimen. An immunotherapy may be a monotherapy or used in combination with one or more other therapies (one or more other immunotherapies or non-immunotherapies).
As used herein, the term “cytotoxic agent” refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells in vitro and/or in vivo. The term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu), chemotherapeutic agents, toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, and antibodies, including fragments and/or variants thereof.
As used herein, the term “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, such as, for example, taxanes, e.g., paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil, vincristine, vinblastine, anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON, GTx, Memphis, Tenn.), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, etc. Examples of anti-cancer agents, including chemotherapeutic agents that may be used in conjunction with the compounds of the invention are listed in Table 2. In a preferred embodiment, the chemotherapeutic agent is one or more anthracyclines. Anthracyclines are a family of chemotherapy drugs that are also antibiotics. The anthracyclines act to prevent cell division by disrupting the structure of the DNA and terminate its function by: (1) intercalating into the base pairs in the DNA minor grooves; and (2) causing free radical damage of the ribose in the DNA. The anthracyclines are frequently used in leukemia therapy. Examples of anthracyclines include daunorubicin (CERUBIDINE), doxorubicin (ADRIAMYCIN, RUBEX), epirubicin (ELLENCE, PHARMORUBICIN), and idarubicin (IDAMYCIN).
Erwinia L-asparaginase
While oncolytic viruses and/or the therapies that induce depletion of tumor-induced PMN-MDSC of the invention can be administered to subjects as isolated agents, it is preferred to administer these viruses or therapies as part of a pharmaceutical composition. Therefore, the subject invention thus further provides compositions comprising a combination of an oncolytic virus, a compound that induces depletion of tumor-induced PMN-MDSC, and at least one pharmaceutically acceptable carrier. The pharmaceutical compositions can be adapted for various routes of administration, such as enteral, parenteral, intravenous, intramuscular, topical, subcutaneous, and so forth. Administration can be continuous or at distinct intervals, as can be determined by a person of ordinary skill in the art. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, and includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
The compositions administered in accordance with the methods of the invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science (Martin, E. W., 1995, Easton Pa., Mack Publishing Company, 19th ed.) describes formulations which can be used in connection with the subject invention. Formulations suitable for administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions of the subject invention can include other agents conventional in the art having regard to the type of formulation in question.
Compositions of the invention, the oncolytic viruses, the therapies that induce depletion of tumor-induced PMN-MDSC, and others agents used in the methods of the invention may be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site, e.g., injected or topically applied to the tumor), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. Compositions of the invention and other agents used in the methods of the invention may be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the agents may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the compositions and agents may be incorporated into sustained-release preparations and devices.
The oncolytic viruses and/or the therapies that induce depletion of tumor-induced PMN-MDSC can be administered into the tumor (intra-tumorally) or into a lymph node, such as inguinal lymph node of the subject. The oncolytic viruses and/or the therapies that induce depletion of tumor-induced PMN-MDSC can also be administered intradermally, intravenously, or intraperitoneally by infusion or injection.
Solutions of the active agents can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the oncolytic viruses and/or the therapies that induce depletion of tumor-induced PMN-MDSC in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the compositions and agents may be applied in pure-form, i.e., when they are liquids. However, it will generally be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the peptide can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Additives such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions which can be used to deliver the peptides to the skin are disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman (U.S. Pat. No. 4,820,508).
Useful dosages of the pharmaceutical compositions of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
Accordingly, the present invention includes a pharmaceutical composition comprising the oncolytic viruses and/or the therapies that induce depletion of tumor-induced PMN-MDSC, optionally, in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for oral, topical or parenteral administration, comprising an oncolytic virus and/or a therapy that induces depletion of tumor-induced PMN-MDSCs constitute a preferred embodiment of the invention. The dose administered to a patient, particularly a human, in the context of the present invention should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition. Advantageously, in some embodiments, administration of the compounds of the invention does not induce weight loss or overt signs of toxicity in the subject.
A suitable dose(s) results in a concentration of the active agent in cancer tissue, such as a malignant tumor, which is known to achieve the desired response. The preferred dosage is the amount which results in maximum inhibition of cancer cell growth, without unmanageable side effects. Administration of the oncolytic viruses and the therapies that induce depletion of tumor-induced PMN-MDSC and optionally, other agents can be continuous or at distinct intervals.
To provide for the administration of such dosages for the desired therapeutic treatment, in some embodiments, pharmaceutical compositions of the invention can comprise between about 0.1% and 45%, and especially, 1 and 15%, by weight of the total of one or more of the agents of the invention based on the weight of the total composition including carrier or diluents. Illustratively, dosage levels of the administered active ingredients can be: intravenous, 0.01 to about 20 mg/kg; intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about 100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about 200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation, 0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal (body) weight.
To facilitate the understanding of the subject matter disclosed herein, a number of terms, abbreviations or other shorthand as used herein are defined below. Any term, abbreviation or shorthand not defined is understood to have the ordinary meaning used by a skilled artisan contemporaneous with the submission of this application.
The term “subject,” as used herein, describes a mammal including, but not limited to, humans, apes, chimpanzees, orangutans, monkeys, dogs, cats, horses, pigs, sheep, goats, mice, rats, and guinea pigs.
The term “treatment” or any grammatical variation thereof (e.g., treat, treating, and treatment etc.), as used herein, includes, but is not limited to, ameliorating or alleviating a symptom of a disease or condition; reducing or delaying recurrence of a condition; reducing, suppressing, inhibiting, lessening, or affecting the progression and/or severity of an undesired physiological change or a diseased condition. For instance, treatment includes, for example, preventing, inhibiting, or slowing the rate of development of a cancer or conversion of a benign cancer into a malignant cancer; slowing the growth and/or proliferation of cancer; and reducing the size or spread of cancer.
The term “effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a cancer or is otherwise capable of producing an intended therapeutic effect. In certain embodiments, the effective amount enables a 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, 99% or 100% reduction in the rate of formation of a tumor or spread of a cancer. In certain embodiments, the effective amount enables a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% reduction in the size of a tumor or the spread of a cancer.
As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” includes more than one such compound. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” The transitional terms/phrases (and any grammatical variations thereof) “comprising,” “comprises,” “comprise,” “consisting essentially of,” “consists essentially of,” “consisting” and “consists” can be used interchangeably.
The phrases “consisting essentially of” or “consists essentially of” indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim.
With respect to an oncolytic virus having a deletion of a gene, the term “deletion” refers to genetic modifications done to the gene including any of the open reading frame, upstream regulatory region and downstream regulatory region that result in down regulation or complete inhibition of the transcription of the open reading frame (ORF) of the gene. Deletion can be achieved either by deleting the entire ORF or a portion of the ORF, for example, by introducing: a frame shift mutation, a missense mutation, a sequence that disrupt the activity of the protein encoded by the gene, a stop codon, or any combination thereof.
With respect to a virus containing a heterologous gene, the term “heterologous gene” includes an open reading frame and can further optionally comprise one or more additional elements of a gene, such as an upstream regulatory region, a downstream regulatory region, and/or a terminator.
All mice were maintained according to approved procedures. 6-8 week-old female BALB/c and C57BL/6N mice were used.
Vero cells, purchased from ATCC, and B16F10 cells, a kind gift, were maintained in complete Dulbecco's modified Eagle's medium (DMEM, Gibco; supplemented with 10% FBS and antibiotics). AB1 cell line, purchased from European Collection of Cell Cultures, was maintained in complete Roswell Park Memorial Institute-1640 medium (RPMI, Gibco; supplemented with 10% FBS, 2 mM L-glutamine and antibiotics). Luciferase-expressing cells were maintained in complete RPMI supplemented with 1 μg/ml puromycin (Invitrogen). T cells and splenocytes were cultured in complete RPMI supplemented with 50 μM 2-mercaptoethanol (Sigma).
A highly attenuated MVTT virus encoding dual reporters of HcRed and HIV-1 p24 was prepared. MVTT viral stocks were prepared and virus titers were determined by plaque forming assay in Vero cells using serially diluted virus. In vitro infection was performed in 24-well plate with 2×105 AB1 mesothelioma cells in each well. 0.2 MOI recombinant MVTT was added into the culture to allow 1 hour attachment before cells were washed and incubated with 1 ml fresh medium. Culture supernatants were harvested 24, 48, and 72 hours after infection, and viral titers were measured by serial dilution and plaque forming assay in Vero cells. Released HMGB1 were examined by western blotting using anti-HMGB1 antibody (Abcam, ab79823). Released ATP in the supernatant and cell viability were determined by CellTiter-Glo luminescent cell viability assay (Promega) per the manufacturer's instructions. Relative cell viability was calculated with ratio of luminescence between infected cells and uninfected cells. Cells were also detached and incubated with anti-CRT antibody (Abcam, ab92516) for surface labelling and flow cytometric analysis. CRT expression in the cell lysates was also determined by western blotting. AB1-MVTT viral supernatant used for antigen-presentation assay was collected 48 hours after infection. Cell debris was removed by centrifugation, passed through a 0.2 μm low-protein binding membrane (Millipore) and heat-inactivated at 60° C. for 1 hour. Successful elimination of live virus was confirmed by plaque forming assay in Vero cells.
Mesothelioma AB1 cells or melanoma B16F10 cells were harvested and single cell suspensions of 5×105 cells in 100 μl PBS were injected s.c. into right hind flank of BALB/c or C57BL/6N mice, respectively. Tumor volumes were measured by caliper and calculated with the formula: Tumor volume=1/2(length×width). Luciferase-expressing tumors were also measured by bioluminescence imaging using an IVIS spectrum (PerkinElmer) and signal intensity was presented as photons/s/cm2/sr within regions of interest (ROI) using Living Image software (version 4.0, PerkinElmer), as previously described. Intra-tumoral treatment of established tumors was started at 7 days after tumor inoculation. Tumors were injected with 100 μl of recombinant MVTT, anti-Ly6G antibody (clone 1A8, BioXCell) or combination of the two. 1A8 was administered at 100 μg per dose and rat IgG2a (clone 2A3, BioXcell) was injected alone or in combination with recombinant MVTT as an isotype control. Mice that rejected tumors were re-challenged with 2×106 tumor cells via an s.c. injection on their opposite flank. All animals were euthanized when tumor length reached more than 15 mm.
Splenocytes were isolated as previously described. Tumors were cut into pieces and digested with 1 mg/ml collagenase IV (Sigma) and 0.5 U/ml Dnase I (Roche) for 1.5 hours at 37° C. Cells were passed through a 70 μm strainer and then subjected to 40%/80% Percoll gradient (Sigma). Leukocytes at the interphase were recovered after centrifuge at 800 g for 20 min. Bone-marrow leukocytes were flushed out from tibia and femur. Cells were then passed through a 70 μm strainer and red blood cells were removed using red blood lysis buffer (BD Biosciences).
Single-cell suspensions of splenocytes were used for cell isolation. CD3+ T cells were isolated using Dynabeads Untouched T Cell Kits (Thermo Scientific). CD4+ and CD8+ T cells were isolated using T Cell Isolation Kit (Miltenyi). Total MDSCs or MDSCs subsets were isolated using MDSCs Isolation Kit (Miltenyi), according to manufacturer's instructions.
Purified MDSCs were labelled with CFSE (Thermo Scientific). 4×106 MDSCs were intravenously injected into AB1 tumor-bearing mice through tail vein. Labelled MDSCs were detected 24 hours after transfer.
CD4+ and CD8+ T cells were depleted during treatment by intraperitoneal injection of 250 μg anti-CD4 (YTS191.1, BioXcell) or anti-CD8 (YTS169.4, BioXcell), respectively, every 5 days, starting 1 day before therapy. Successful T cell depletion was confirmed by flow cytometric analysis of peripheral blood mononuclear cell (PBMC). Anti-Ly6G (clone 1A8) and corresponding isotype (clone 2A3) were also purchased from BioXcell.
Cytokine concentrations in the culture supernatant were measured by LEGENDplex T Helper Cytokine Panel (BioLegend). Tumors were cut into pieces and homogenized in T-PER Tissue Protein Extraction Reagent (Thermo Scientific) supplemented with Protease Inhibitor Cocktail (Roche). Chemokine concentrations were determined by LEGENDplex Proinflammatory Chemokine Panel (BioLegend) and normalized against total proteins determined by BCA protein assay (Thermo Scientific).
Following a standard protocol, isolated bone-marrow cells were plated in 6-well plate at 3×106 cell per well in the presence of 40 ng/ml GM-CSF and IL-4. Half of the differentiation medium was replaced every 2 days. On day 9, loosely adherent cells were resuspended by repeated pipetting and collected together with non-adherent cells in the supernatant for flow cytometric analysis with surface staining of anti-CD3, anti-CD11c and anti-WIC II, resulting in >90% CD11c+MHC II+ BMDCs. For BMDCs-T cells co-culture, BMDCs were pooled and seeded into 96-well V-bottom plate at 2×104 cells per well in the presence of 100 μl inactivated AB1-MVTT viral supernatant or culture medium. In some cultures, anti-CRT antibody (Abcam, ab92516) or rabbit IgG was added at 100 ng/ml. After incubation overnight, BMDCs were thoroughly washed with culture medium and CFSE labelled CD3+ T cells were added at a ratio of 1:1, for an additional culture of 10 days, with replacement of half of the culture medium every 4 days. Culture supernatant collected on day 7 and cells collected on day 10 were subjected to analysis of cytokine secretion and T cell proliferation, respectively. For BMDCs-MDSCs co-culture, BMDCs were seeded in 96-well U-bottom plate at 5×104 cells per well, stimulated by 100 ng/ml LPS (Sigma) or 100 μl inactivated AB1-MVTT viral supernatant, in the presence of purified PMN-MDSCs or M-MDSCs. To clearly distinguish BMDCs from MDSCs by flow cytometry, purified MDSCs subsets were labelled with CFSE prior to incubation with BMDCs. 48 hours after LPS-stimulation, BMDCs maturation was assessed via flow cytometry. When cells were stimulated with AB1-MVTT viral supernatant, half of the medium was replaced with fresh culture medium on day 4 and supernatant was collected on day 7 to assess cytokine secretion.
BMDCs were seeded in 96-well U-bottom plate at 5×104 cells per well and were subjected to incubate with 5 μg/ml anti-mouse CD210 (IL-10R, clone 1B1.3a, BioLegend) antibody for 30 min at 37° C. Then 1×105 CFSE labelled PMN-MDSCs or M-MDSCs were added into the culture at a ratio of 2:1 with BMDCs, following stimulation with 100 ng/ml LPS for 48 hours in the incubator. Culture volume was maintained at 100 p1 each well and rat IgG1 (eBioscience) was used as isotype control.
Cell surface and intracellular immunostaining were performed as previously described. The following antibodies were purchased from eBioscience: anti-CD11b (clone M1/70), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8-Ly6 g), anti-CD3 (clone 17A2), anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), anti-PD1 (clone J43), anti-Tim3 (clone RMT3-23), anti-CD11c (clone N418), anti-MHC II (clone M5/114.15.2), anti-CD80 (clone 16-10A1), and anti-CD49b (clone DX5). The following antibodies were purchased from BioLegend: anti-CD25 (clone 3C7), anti-Foxp3 (clone 150D), anti-CXCR2 (clone SA045E1), and anti-CXCR3 (clone CXCR3-173). Anti-CCR2 (clone REA538) antibody was purchased from Miltenyi. Samples were run on a BD FACSAria II cell sorter (BD Biosciences) and analyzed using FlowJo (Tree Star, v10).
IFN-γ-producing T cells in isolated splenocytes were assessed by ELISpot assay. gp70-AH1 (SPSYVYHQF), OVA257-264(SIINFEKL), GP100 (EGPRNQDWL), TRP2 (SVYDFFVWL), and TWIST1 peptides (15-mers spanning the entire amino acid sequence with 11 amino acids overlapping) were synthesized by GL Biochem (Shanghai). Cytotoxic effect of purified T cells against AB1 cells was determined using LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Scientific), as previously described.
All data are presented as mean±s.e.m. Significance was determined by the two-tailed Student t-test and p-value <0.05 was considered statistically significant. Survival of all animals was plotted on Kaplan-Meier survival curve and the log-rank test was performed to analyze differences in GraphPad Prism 5 software.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
To determine the oncolytic effects of MVTT, a recombinant MVTT (rMVTT) was generated to simultaneously express two detection markers, HIV-1 p24 and far-red fluorescent mutant HcRed (
To investigate the ability of rMVTT in treatment of established AB1 mesothelioma in Balb/c mice, the i.t. viral injection was explored as a means to determine its direct antitumor efficacy. Mice were inoculated with AB1 mesothelioma cells 7 days before they received different doses of rMVTT treatment, classified as high-, medium-, low-dose groups (
Because the initiation of adaptive antitumor immunity after oncolysis primarily occurs inside the tumor, the TME after rMVTT treatment was examined. At two time-points, 2 and 4 days, after intra-tumoral rMVTT treatment, different tumor resident immune cells, including proportions of CD3+ T cells, natural killer (NK) cells, CD4+ Treg (CD4+ CD25+Foxp3+) and MDSC subsets (PMN-MDSC, CD11b+Ly6G+Ly6Clow/int; M-MDSC, CD11b+Ly6G-Ly6Chi), and expression of the exhaustion surface markers PD-1 and Tim-3 on CD3+ T cells were measured. MDSCs and Tregs are major components of the tumor suppressive microenvironment. The overall levels of MDSCs found in the spleen appeared to decrease over time after rMVTT treatment, while frequencies of tumor-infiltrating MDSCs were maintained at similar levels (
To understand how PMN-MDSCs were recruited into tumors, the role of chemokine induced by rMVTT treatment was examined. Flow cytometric analysis of chemokine receptors revealed that CXCR2 was expressed only on PMN-MDSCs but not on M-MDSCs. Conversely, high level of CCR2 expression was found on M-MDSCs but not on PMN-MDSCs (
To prevent the migration of MDSCs into tumors, the efficacy of a MDSC depleting antibody, the anti-Ly6G monoclonal antibody 1A8, was tested. Since 1A8 is routinely used to deplete Ly6G+ MDSCs, AB1 tumor-bearing mice were treated via the i.t. route with 1A8 or isotype control. Compared with the isotype control, the 1A8-treated mice had significantly decreased frequency of splenic MDSCs yet this antibody did not show efficacy in reducing total MDSCs accumulation in tumors. As expected, however, 1A8 selectively diminished Ly6G+ PMN-MDSCs in both spleen and tumor at day-2 after the injection (
Considering that MDSCs are one of the major types of immunosuppressive cells that inhibit antitumor T cell responses, whether the prevention of MVTT-induced tumor trafficking of PMN-MDSCs would enhance the therapeutic efficacy of the oncolytic viral treatment was examined. In a similar setting as described above, Balb/c mice bearing 7-day-old AB1 tumors were simultaneously injected with rMVTT plus either 1A8 or isotype control. To improve antitumor effect, an additional combination treatment was given 2 days later (
Thus, PMN-MDSCs depletion could largely improve the effects of the rMVTT treatment probably by inducing prolonged antitumor immunity. To test this, tumor-specific T cell responses were measured. Murine splenocytes were harvested and tested against tumor antigen either gp70-AH1 or TWIST1 peptides. Significantly increased T cell responses against both gp70-AH1 and TWIST1 were elicited among mice treated with the rMVTT+1A8 combination (
As noted above, MVTT-induced oncolysis of tumors created an immune activating environment with the production of CRT, HMGB1, and ATP. Yet dendritic cells (DCs) failed to recognize and integrate these signals to drive T cell activation. The presence of PMN-MDSCs may supress DC function during MVTT-induced oncolysis of tumors. To test this, the direct impact of PMN-MDSCs on DCs was determined. The ability of bone-marrow derived DCs (BMDCs) in processing and presenting antigens for activating CD3+ T cells derived from controller mice that received the MVTT+1A8 combination treatment was determined. MVTT-infected AB1 cell supernatant as a source of tumor antigen pool was used to pulse BMDCs. Antigen-loaded BMDCs greatly enhanced the production of TNF-α and IFN-γ (
Subsequently, direct interaction between AB1-induced MDSCs and BMDCs was measured with either culture medium alone or LPS as a maturation signal. As expected, LPS itself significantly increased the level of CD80 expression on BMDCs (P<0.0001, Med vs. LPS) (
In addition, the effectiveness of the combination therapy was also confirmed in a distinct syngeneic C57BL/6 melanoma model, where enhanced B16F10 tumor regression, prolonged survival and augmented antitumor T cell responses (
Because the initiation of adaptive antitumor immunity after oncolysis primarily occurs inside the tumor, the TME was examined after rMVTT treatment. Analysis of rMVTT-injected AB1 mesothelioma revealed that expression of virus-encoded HcRed was readily detected 2 days after intra-tumoral injection and rapidly decreased thereafter (
To examine whether PMN-MDSCs may preferentially be recruited to the TME after rMVTT treatment, the expression of chemokine receptors on both MDSC subsets and the levels of chemokines in rMVTT-treated tumors were examined. Flow cytometric analysis of chemokine receptor expression revealed that CXCR2 was expressed only on PMN-MDSCs but not on M-MDSCs. Conversely, high levels of CCR2 expression were found on M-MDSCs but not on PMN-MDSCs (
To investigate the role of MDSCs in the rMVTT treatment, two MDSC-depleting agents, anti-Ly6G monoclonal antibody 1A8 and the specific depleting peptibody H6-pep, were explored in our mesothelioma model. 1A8 is routinely used to deplete Ly6G+ cells, primarily PMN-MDSCs, whereas H6-pep and G3-pep are two peptibodies with binding specificity to both PMN-MDSCs and M-MDSCs. Accordingly, these two peptibodies were manufactured by a transient expression system in 293F cells using expression plasmids (
The efficacy of 1A8 and H6-pep during rMVTT treatment was then studied. rMVTT treatment resulted in the increased recruitment of PMN-MDSCs in tumors (
Considering that MDSCs are one of the major immunosuppressive cells that inhibit antitumor T cell responses, whether the depletion of PMN-MDSCs enhanced the therapeutic efficacy of MVTT-based oncolytic virotherapy was explored. In a similar setting as described above, BALB/c mice bearing 7-day-old wild-type AB1 mesothelioma were simultaneously injected with low-dose rMVTT (1×107 PFU) in combination with either 100 μg of 1A8 or H6-pep for the specific depletion of PMN-MDSCs and M-MDSCs, respectively (
To further test this hypothesis, tumor-reactive T cell responses were measured. Murine splenocytes were harvested and tested against gp70-AH1 or TWIST1 peptides (
Although rMVTT-induced oncolysis created an immune-activating environment with the production of CRT, HMGB1 and ATP, anti-mesothelioma specific T cell responses were not readily induced (
Subsequently, the direct interaction between AB1-induced MDSCs and BMDCs was measured. BMDCs were co-cultured with AB1-induced MDSCs in the presence or absence of LPS. CD80 and CD86 expression on BMDCs was significantly upregulated by LPS stimulation (P<0.001 for CD80, P<0.05 for CD86, Unstimulated versus LPS), suggesting BMDC maturation (
To understand the underlying mechanism of PMN-MDSC-mediated immunosuppression, productions of IL-10 and TGF-β in MDSC subsets were examined. MDSCs did not produce TGF-β and only PMN-MDSCs exhibited an IL-10-producing subset (
This application claims the benefit of U.S. Provisional Patent Application Ser. Nos. 62/660,546, filed Apr. 20, 2018, and 62/687,531, filed Jun. 20, 2018, which are hereby incorporated by reference in their entirety including any tables, figures, or drawings.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2019/000525 | 4/19/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62660546 | Apr 2018 | US | |
| 62687531 | Jun 2018 | US |
